PhD Students’ works
Progress Report 2010
Oncology and Genetics
Doctoral School
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University of Siena
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R
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F
PhD Students
works
Progress Report 2010
Oncology and Genetics
Doctoral School
Molecular Biology Department
and
Human Pathology and Oncology Department
Information Engineering Department
Pediatrics, Obstetrics and Reproduction Medicine Department
Surgery Department
Surgery and Bioengineering Department
and
I.T.T. Istituto Toscano Tumori
S.H.R.O. Sbarro Health Research Organization
Copyright 2010
Impaginazione: Ufficio Comunicazione Online
Stampa: Centro Stampa di Ateneo
Finito di stampare 7 settembre 2010
Oncology and Genetics Doctoral School
This initiative is aimed to spread the information on the research activities of PhD
students in our academic community.
The pamphlet is in English in order to promote Doctoral Schools of our University at
international level, with particular attention to those foreign institutions with which we
have signed international cooperation agreements. Moreover, it could also be useful to foster new agreements with foreign partners.
The Rector
Prof. Silvano Focardi
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Oncology and Genetics Doctoral School
This pamphlet was created to regroup and present together the research activities of the
students of the Doctoral School in Oncology and Genetics in order to spread information
about the work of the students and to promote the collaboration on research projects.
The first pages illustrate the activity of the “annual progress report day”. This event takes
place at the end of each academic year and is dedicated to the presentation of both the
research projects proposed by the new entered students and the annual progress reports
of the older students. The pamphlet continues with the presentation of the research
abstracts of the 35 PhD students. Finally, the last pages are dedicated to the “thesis discussion days” and qualification of “Doctor Europaeus”.
I wish to dedicate this pamphlet to the PhD students who represent the “mainstay” of the
Institution that we call University with their continuous daily work, their perseverance and
motivation.
The director of the School
Prof. Alessandra Renieri
-5-
Oncology and Genetics Doctoral School
The Doctoral School in Oncology and Genetics is constituted of 5 sections or “education trainings”:
1) Medical Genetics coordinated by Alessandra Renieri
2) Oncological Genetics coordinated by Antonio Giordano
3) Colorectal and Gastroesophageal Diseases coordinated by Gabriello Tanzini
4) Hepatobiliopancreatic Diseases and Multitumoral Syndromes coordinated by Francesco Cetta
5) Bioinformatics coordinated by Monica Bianchini
In addition to the five above mentioned coordinators, the Faculty Board is composed by teachers from
the University of Siena: Francesca Ariani, Alessandro Cappelli, Anton Ferdinando Carli, Maddalena
Cioni, Serenella Civitelli, Paolo Frezzotti, Theodora Hadjistilianou, Marco Lorenzi, Francesca Mari,
Giuseppe Marzocca, Clelia Daniela Anna Miracco, Marco Mugnaini, Roberto Ponchietti, Maria Lucia
Sampoli, Franco Scarselli, Francesco Tani, Walter Testi, Paolo Toti, Luigi Verre; and by teachers from
other Universities: Pier Paolo Pandolfi from the Cornell University, New York, Hans van Bokhoven
from the University of Nijmegen, The Netherlands.
The following additional teachers from the University of Siena compose the Council of the School:
Alfio Andronico, Maurizio Botta, Mirella Bruttini, Ilaria Cardinali, Mario Carmellini, Mario Chiarello,
Concetta Gardi, Maurizio Genuardi, Marco Maggini, Carmela Marinelli, Domenico Mastrangelo,
Alessandro Piccolomini, Paola Piomboni, Enrico Pinto, Franco Roviello, Edmondo Trentin, Claudia
Torricelli.
On the basis of research activity the School has signed 7 International Cooperation Agreements with
the following Universities:
Bilkent University, Ankara, Turkey;
Duisburg-Essen University, Germany;
Freiburg University, Germany;
Greenwood Genetic Center, Greenwood, South Carolina, USA;
Kentucky University, Lexington, USA;
Radboud University of Nijmegen, The Netherlands;
St. Kliment Ochridski University, Sofia, Bulgaria.
The Doctoral School in Oncology and Genetics at the University of Siena trains students to carry out
research in Medical Genetics and in Clinical and Molecular Oncology over a four years program. The
aim of this Doctoral School is to train researchers who will be able to plan and develop competitive
research proposals. The School has a dedicated web site at the following address:
http://www.unisi.it/ricerca/dottorationweb/genetica_medica/. In this site it is possible to find general
information on the School, seminar activities, research projects, and PhD students scientific “identity
card”.
The School on the basis of the high quality of the education activities and the internationalization of
the scientific and teaching courses has been selected by a external board as one of the PhD Schools
of the University of Siena belonging to the Graduate College Santa Chiara. The Doctoral Schools of
the Graduate College join in multidisciplinary and international research projects, creating a centre of
high qualification for postgraduate education. The PhD students of the Graduate College are called
“santachiarini” and are provided with the additional title of the Graduate College and the stay in the
University residences. Residences of the Graduate College are situated in the old town. In these buildings teaching activities, conferences and interdisciplinary courses and seminars take place, but the
most innovative aspect is that they are informal places for meetings where PhD students and teachers can stay and eat together.
-7-
Index
Annual progress report day
Pag. 11
Students Project Abstracts
Pag. 15
Thesis Discussion
Pag. 53
• Giovanni Abbadessa
A phase 1 dose escalation study of ARQ 197 in adult patients with metastatic solid
tumors
Pag. 17
• Mariangela Amenduni
Induced pluripotent stem cells as a human model to study Rett syndrome
Pag. 18
• Annamaria Azzarà
Environmental Pollution in Metropolitan Areas and the occurrence of respiratory
dysfunction and respiratory diseases
Pag. 19
• Simona Benoni
Increased prevalence of neoplasms in subjects with progeroid syndromes: a genotypephenotype correlation in a personal series and in patients from the literature
Pag. 20
Pag. 21
• Maria Grazia Bruccheri
Study of genetic susceptibility factors for Multiple Chemical Sensitivity (MCS) in sicilian
patients
• Antonella Chessa
Endothelin 1 and endothelin-converting enzyme in primary colorectal cancer
Pag. 22
• Filomena Cisternino
Genetics and molecular biology in the multidisciplinary approach to pressure
ulcers and lower limb ulcers in diabetics
Pag. 23
• David Colecchia
Multiple primary malignancies: Yeta challange
Pag. 24
• Daniele Conti
Identification of the regulatory mechanisms of Cdk2/CyclinA inhibition by pRb2/p130
protein
Pag. 25
• Martina Cozzi
New small molecule inhibitors of Src as potential candidates for cancer therapy
Pag. 26
• Francesca Crucianelli
Detection of constitutional epigenetic changes in multiple cancers by MS-MLPA analysis
Pag. 27
• Roberta De Filippis
FOXG1 mutation leading to reduced chromatin affinity causes “Rett fruste” overlapping
with EHMT1 phenotype
Pag. 28
Pag. 29
• Vittoria Disciglio
Role of MDM2 T309G and TP53 R72P polymorphisms in modulation of variable phenotypic expression of retinoblastoma
• Andrea Fontani
Outcome of surgical treatment of colorectal cancer in the elderly
Pag. 30
• Iris Maria Forte
Gastric cancer and cell cycle regulation
Pag. 31
• Elisa Grillo
European network database on Rett syndrome
Pag. 32
• Valeria Guarnaccia
Retinoic acid and breast cancer: how to improve the therapeutic effect on the basis
of molecular knowledge
Pag. 33
-8-
Pag. 34
• Valentina Guercio
PM chemical characterization and differences in cytotoxicity versus pro-inflammatory potency
of different PM fractions in human epithelial lung cells
• Baharak Khadang
New Strategies for the Study and Treatment of Malignant Mesothelioma
Pag. 35
• Raffaele La Montagna
Androgen receptor and PIN1 in prostate cancer
Pag. 36
Pag. 37
• Paolo Laviano
Study of obesity prevalence, body mass index including energy consumption measured by
accelerometer and pollution related health effects in children attending primary school in Milan
• Gabriella Livide
Mutational screening in the RB1 gene
Pag. 38
• Bruno Lorenzi
Evaluation of anorectal function after radiotherapy in patients treated for rectal cancer
Pag. 39
• Giuliana Malagnino
Intrinsic toxicity, inflammatory potency and individual susceptibility in the occurrence
of health damage from Particulate Material (PM)
Pag. 40
• Elena Marcocci
Autosomal Alport syndrome: a new model including both dominant and recessive
inheritance
Pag. 41
• Monica Mischitelli
Prostate cancer: the influence of STAT3 and the presence of Human Polyomavirus BK in cells
Pag. 42
• Mafalda Mucciolo
Copy number variations analysis in autism spectrum disorders
Pag. 43
• Olabinjo Olayinka
Bioinformatics approach to the pRb pathway in cancer initiation and progression
Pag. 44
• Marco Pacifici
Detection and role of mir146a in HIV-clinical samples
Pag. 45
• Filomena Tiziana Papa
Diagnosis process assessment of Array-CGH analysis
Pag. 46
• Veronica Parri
Identification and characterization of deletions and duplications by MLPA in patients
with Cohen syndrome
Pag. 47
• Flavio Rizzolio
Pin1 controls cell cycle progression through interaction with pRB
Pag. 48
• Dalila Rondinella
QF-PCR as a tool for rapid prenatal diagnosis
Pag. 49
• Ariele Spanhol Rosseto
Molecular work-up of patients with the congenital variant of Rett syndrome: analysis
of the 14q12 region.
Pag. 50
• Rosalia Zangari
The impact of traffic pollution on antioxidant system of two populations exposed to
different levels of pollutants
Pag. 51
-9-
Annual Progress Report
Oncology and Genetics Doctoral School
September 7, 2010 Centro Didattico S Maria alle Scotte, room 13
8.45
Welcome Addresses
Silvano Focardi, Rector of the University of Siena
Alessandra Renieri, Director of Oncology and Genetics Doctoral School
9.00
Progress report of the 3rd year, XXIII cycle (10 minutes for each one)
Chairmen: Alessandra Renieri and Francesco Cetta
Amenduni Mariangela (A. Renieri)
Induced pluripotent stem cells as a human model to study Rett syndrome
Azzarà Annamaria (F. Cetta)
Environmental Pollution in Metropolitan Areas and the occurrence of respiratory dysfunction and diseases
De Filippis Roberta (A. Renieri)
FOXG1 mutation leading to reduced chromatin affinity causes “Rett fruste” overlapping
with EHMT1 phenotype
La Montagna Raffaele (A. Giordano)
Androgen receptor and PIN1 in prostate cancer .
Laviano Paolo (F. Cetta)
Study of obesity prevalence, body mass index including energy consumption measured
by accelerometer and pollution related health effects in children attending primary school
in Milan
Mischitelli Monica (A. Giordano- VA. Pietropaolo)
Prostate cancer: the influence of STAT3 and the presence of Human Polyomavirus BK in cells
Parri Veronica (A. Renieri)
Identification and characterization of deletions and duplications by MLPA in patients
with Cohen syndrome
Rondinella Dalila (A. Renieri)
QF-PCR as a tool for rapid prenatal diagnosis
10.30 Progress report of the 2nd year, XXIV cycle (10 minutes for each one)
Chairman: Monica Bianchini
Bruccheri Maria Grazia (A. Renieri)
Study of genetic susceptibility factors for Multiple Chemical Sensitivity (MCS)
in sicilian patients
Colecchia David (M. Chiariello)
Involvement of the Erk8 in autophagy
Conti Daniele (A. Giordano)
Identification of the regulatory mechanisms of Cdk2/CyclinA inhibition by pRb2/p130 protein
Crucianelli Francesca (G. Tanzini - M. Genuardi)
Detection of constitutional epigenetic changes in multiple cancers by MS-MLPA analysis
Disciglio Vittoria (A Renieri)
Role of MDM2 T309G and TP53 R72P polymorphisms in modulation of variable phenotypic expression of
retinoblastoma
Forte Iris Maria (A. Giordano)
Gastric cancer and cell cycle regulation
- 11 -
Oncology and Genetics Doctoral School
Mucciolo Mafalda (A. Renieri)
Copy number variations analysis in autism spectrum disorders
Pacifici Marco (A. Giordano)
Detection and role of mir146a in HIV-clinical samples
Zangari Rosalia (F. Cetta)
The impact of traffic pollution on antioxidant system of two populations exposed to different
levels of pollutants
12.30
Finger social lunch and poster viewing
AFTERNOON
SECTION
14.00 Progress report of the 1st year, XXV cycle (5 minutes for each one)
Chairmen: Mario Chiariello e Serenella Civitelli
Cozzi Martina (A. Giordano)
New small molecule inhibitors of Src as potential candidates for cancer therapy
Fontani Andrea (G. Tanzini)
Outcome of surgical treatment of colorectal cancer in the elderly
Grillo Elisa (A. Renieri)
European Rett database network
Guercio Valentina (F. Cetta)
PM chemical characterization and differences in cytotoxicity versus pro-inflammatory
potency of different PM fractions in human epithelial lung cells
Livide Gabriella (A. Renieri)
Mutational screening in the RB1 gene
Olabinjo Olayinka (A. Giordano)
Bioinformatics approach to the pRb pathway in cancer initiation and progression
15.00
Presentation of the PhD students program of the XXVI cycle
16.00
Closing session and attribution of credits by the faculty board
A copy of the minutes is available at http://www.unisi.it/ricerca/dottorationweb/genetica_medica/ accessing
the "Minutes" link.
- 12 -
Oncology and Genetics Doctoral School
- 13 -
Students Project Abstracts
Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Oncological Genetics
XXI cycle
Giovanni Abbadessa, MD
[email protected]
Tutor A. Giordano
A phase 1 dose escalation study of ARQ 197 in adult patients with metastatic solid
tumors
ARQ 197, an oral selective c-MET inhibitor, and has effects on the tumour in preclinical models. Based on its
pharmacologic effect, it may be a useful cancer treatment. This study was performed to determine the maximum tolerated dose (MTD) of ARQ 197 in patients with advanced, refractory metastatic or recurrent solid
tumours. Dose-limiting toxicities (DLTs), safety, pharmacokinetic parameters and tumour response were also
evaluated.
In this open-label, Phase I, dose escalation study ARQ 197 was administered orally initially in repeated cycles
of 21 days (14 days on/7 days off treatment) and later modified to continuous dosing. Thirteen dose levels
and 2 formulations were investigated: from 10 mg to 360 mg twice daily (bid). Treatment continued until unacceptable toxicity, tumour progression or death.
Seventy-four patients were treated in this trial. No MTD was determined in this study. ARQ 197 was well tolerated, with mild to moderate toxicities. DLTs consisted of Neutropenia in 1 patient and dehydration and vomiting in 1 patient, both at the 360 mg bid dose level. Sixty-one patients were evaluable for assessment of antitumor
activity; 3 patients with neuroendocrine, prostate, and testicular cancers achieved a partial response (PR); 38
patients had a best response of stable disease and 20 had progressive disease, the median duration of therapy being 6.5 weeks (range; 1-119 weeks). With an observed half-life of 2-4 hours, ARQ 197 showed no relevant accumulation upon multiple dosing. Increases in exposure were less than proportional to increasing
dose, and all patients remained above the minimum inhibitory concentration of 0.3 μM for 8 hours or longer.
Results indicate that further clinical investigation of ARQ 197 is warranted, and suggest it could be a promising future therapy for patients with cancer. More Phase 1 and 2 studies have been conducted and are
ongoing, having shown a favourable safety profile as single agent and in combination with agents such as
erlotinib, sorafenib, gemcitabine, biologic activity on tumor cMET from pre- and post- therapy biopsies in
patients, clinical activity in different tumor types.
This work is reported in: Mekhail T et al. J Clin Oncol 2009;27; Abstract 3548.
This work is funded by: ArQule, Inc.
- 17 -
Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Medical Genetics
XXIII cycle
Mariangela Amenduni, BS
[email protected]
Tutor A. Renieri
Induced pluripotent stem cells as a human model to study Rett syndrome
Rett syndrome (RTT) is a severe neurodevelopmental disorder representing one of the most common genetic causes of mental retardation in girls. The classic form is mainly due to MECP2 mutations, while alterations
in CDKL5 and FOXG1 have been identified in RTT variants. Despite extensive efforts, the molecular mechanisms underlying the syndrome remain unclear, mainly due to the lack of satisfactory human cellular models.
To overcome this obstacle, we employed the approach of genetic reprogramming that allows to generate
induced pluripotent stem (iPS) cells directly from patients fibroblasts. We reprogrammed fibroblasts from 2
CDKL5 patients (a male with p.T288I and a female with p.Q347X) and obtained an iPS line with a p.R306C
MECP2 mutation from the collaboration with James Ellis (Toronto) and one iPS line with a p.W255X FOXG1
mutation from the collaboration with Vania Broccoli (Milan). Pluripotency and self renewal potential of reprogrammed clones have been confirmed. We are now setting up the procedure for in vitro neuronal differentiation. The resulting neurons will offer the opportunity to study disease mechanisms directly on the primarily
affected cells. In particular gene expression profile of some genes, whose expression was found altered in
MECP2 mouse models (Fkbp5, Mobp, Plagl1, Ddc, Mllt2h, Eya2, S100a9) will be assessed in both MECP2,
CDKL5 and FOXG1 mutated neurons, in the hypothesis of a common pathway. Particular attention will be
paid to S100a9, altered also in post-mortem RTT brains. These experiments will enable disease investigation
and might allow the identification of potential “druggable” targets for therapeutic approaches.
This work is reported in: Ariani et al. Induced pluripotent stem cells as a human model to study disease mechanisms in Rett
syndrome. 2nd European Congress on Rett Syndrome , 7 – 10 October 2010 - Edinburgh
This work is funded by: Telethon grants GGP09117 and GTB07001C to A.R.
- 18 -
Oncology and Genetics Doctoral School
Doctoral School of Oncology and Genetics
Hepatobiliopancreatic Disease and Multitumural Syndromes
XXIII cycle
Annamaria Azzarà, MS
[email protected]
Tutor Prof. F. Cetta
Environmental Pollution in Metropolitan Areas and the occurrence of respiratory
dysfunction and respiratory diseases
Environmental pollution, mainly due to urban traffic, is responsible for a different air quality in metropolitan
areas vs remote sites and is likely to be responsible for a different incidence of respiratory complications
and/or reduction of pulmonary function.
Two groups of 99 subjects were recruited (n=198), on a casual and voluntary basis: the former in Milan, Italy,
i.e. a densely populated and polluted metropolitan area, the latter in Aprica, a remote alpine site (1181
m.a.s.l), with low pollution, due to traffic or other pollution sources. PM10 and PM2,5 were measured by PM
detection units during two 2 week- campaigns. Each group was classified in to 2 subgroups. The former, aged
30 to 64 years, (n=72) the latter over 65 (n=27).
The following results were obtained: FEV1 (index of bronchial patency) was <80% in 20 out of 99 subjects in
Milan (20,2%), whereas it was < 80% in only 8 out of 99 in Aprica (8,08%).
Evident differences were also observed in subgroups of different age, FEV1 resulting <80% in 8 out of 72
subjects under 65 in Milan (11,1%) and in 2 out of 72 in Aprica (2,8%) and in 12 out of 27 (44,4%) and 6 out
of 27 (22,2%) in subjects over 65, respectively (p<0,05).
Present data, even if preliminary, suggest that, in the polluted metropolitan area of Milan, inhabitants are more
prone to the occurrence of respiratory dysfunction than in the remote alpine site of Aprica (p=0,001). In particular, not only in severely symptomatic subjects with evident respiratory diseases, but also in clinically
asymptomatic individuals, it is possible to detect significant differences in respiratory function because of different levels of environmental pollution.
- 19 -
Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Hepatobiliopancreatic Diseases and Multitumoral Syndromes
XXII cycle
Simona Benoni, BS
[email protected]
Tutor F. Cetta
Increased prevalence of neoplasms in subjects with progeroid syndromes: a genotype-phenotype correlation in a personal series and in patients from the literature.
Most of diseases related to the DNA repair systems deficiency show varying degrees of "accelerated aging"
or cancer. Alterations of ReqQ helicase genes (WRN, BLM, RECQL4), encoding proteins involved in various
types of DNA repair, determine the onset of progeroid syndromes, autosomic recessive diseases, characterized by chromosomal abnormalities, premature aging and high incidence of rare or multiple neoplasms. It is
presumed that the loss of these proteins leads to limited replicative capacity, telomere instability, so to premature cellular senescence and predisposition to cancer.
The aim of this study has been to assess the number and types of cancers concomitantly present in individuals with germ-line mutations in ReqQ genes, and to detect possible genotype-phenotype correlations in this
cohort of patients.
It included an exhaustive review of the literature, the observation of 2 additional Werner patients in Siena and
a cross-sectional approach in a geriatric hospital in Milan, to find similar cases.
No new case of Werner syndrome with associated neoplasms has been detected in our series.
However, at least 17 subjects with mutated WRN gene and with different types of cancer have been found in
the literature. The most frequent associated resulted osteosarcomas, soft-tissue sarcomas and melanomas.
It is likely that the accelerated aging process in WS patients contributes to the higher incidence of rare
tumours, like as soft-tissue sarcomas and other non epithelial malignancies.
- 20 -
Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Medical Genetics
XXIV cycle
Maria Grazia Bruccheri, MD
[email protected]
Tutor A. Renieri
Study of genetic susceptibility factors for Multiple Chemical Sensitivity (MCS) in sicilian patients
Multiple chemical sensitivity (MCS) is a "chronic, recurring disease caused by a person's inability to tolerate
an environmental chemical or class of foreign chemicals". Various theories have been proposed as a cause
of the MCS syndrome including immunologic, genetic, toxicologic, psychologic and sociologic theories. MCS
causes negative health effects in multiple organ systems, like respiratory distress, recurrent infections, seizures, cognitive dysfunction, heart arrhythmia, nausea, headache, and fatigue. The purpose of this study was
to investigate genetic and immunological mechanism, in particular we studied genetic susceptibility, especially genes of importance to the metabolism of xenobiotic compounds. MCS patients were genotyped for
polymorphism in the genes encoding cytochrome P450 (CYP2C9, CYP2C19, CYP2D6) in order to explain
the adverse reactions to drug observed in our patients. We found an apparent association between number
of active cytochrome P450 alleles and MCS status but we could extend this analisys to healthy controls to
confirm the susceptibility. So we can conclude that polymorphisms in several genes contribute to interindividual differences in the metabolism of xenobiotics, and may lead to toxicity and disease. The balance between
activation and/or detoxification processes may influence an individual's susceptibility to diseases.
Polymorphisms cytochrome P450 are not sufficient to cause the MCS phenotype, but further data is needed
before reaching a definitive conclusion.
This work is reported in:
- M. G. Bruccheri et al. Update on Multiple Chemical Sensitivity:from clinical findings to genes using clinical, biochemical,
immunological and genetic approaches. I National congress On MCS, Acireale, 17 April 2010 ( as platform presentations )
- M. G. Bruccheri et al . Analisys of Oxidative Burst activity in 20 patients affected by Multiple Chemical. 42° National congress
of SIBIOC (Società Italiana di Biochimica Cl inica e Biologia Molecolare Cl inica). Roma , 5-8 October 2010 (as poster)
This work is funded by:
IRMA (Istituto di Ricerca Medica e Ambientale)
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Colorectal and Gastroesophageal Diseases
XXI cycle
Antonella Chessa, MD
[email protected]
Tutor Prof. G. Tanzini
Endothelin 1 and endothelin-converting enzyme in primary colorectal cancer
Endothelin 1 ( ET1) is a peptide produced by different human cancer cell lines. HT29 and COLO320 are colon
carcinoma cell lines producing endothelin 1.Elevated plasma levels of ET1 have been demonstrated in
patients with primary colorectal cancer (CRC) with and without liver metastases compared to controls.There
is increasing evidence that ET1 may play a role in the development of CRC both locally and
systemically.Mature ET1,produced mainly by specific endothelin converting enzyme (ECE1) exerts its biological effect via ETA and ETB receptors.Recent results demonstrated that ETA receptors are overexpressed
while ETB are underexpressed in CRC tissue compared to normal colon. In addition application of ETA antagonist via portal vein at the time of tumor implantation reduces subsequent hepatic involvement in the rat
model of colorectal liver metastases.Therefore we investigated the expression and distribution of proteins and
mRNAs of all components of the ET1 system in CRC.Preliminary results by immunoistochemistry show strong
expression of ET1 in stroma and blood vessels of CRC specimens and weak positivity in the normal
colon.Intracellular staining for ECE1 was located in the perinuclear region,probably the Golgi of colonic epithelial cells with eesentially the same distribution in neoplastic and normal tissues.ECE1 inhibitors and endothelin receptors antagonists are indicated as potential anti-cancer agents.Thus knowledge of ECE1 precise
subcellular localization is crucial for effective drug design and delivery.
- 22 -
Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Hepatobiliopancreatic Disease and Multitumoral Syndromes
XXII cycle
Filomena Cisternino, CF
[email protected]
Tutor F. Cetta
Genetics and molecular biology in the multidisciplinary approach to pressure ulcers
and lower limb ulcers in diabetics
Pressure ulcers in the sacral region and ulcers of lower limbs in subjects affected by diabetes-are frequent
(up to 15% of diabetic subjects) in particular in old, frail subjects, with multidistrict imbalance and /or prolonged bed stay.
Recent advance in genetics and molecular biology has shown that hyperexpression of c-myc and endonuclear localization of Beta- catenin, together with abnormal localization of EGFR are typical findings of non
healing ulcers. Analysis using microarray technology of specimens from non healing ulcers have shown significant differences in comparison to specimens that undergo easy healing.
Thanks to a cooperation with the Dept of Special Surgery of the N:Y University (Prof. H. Brem), a multidisciplinary approach to pressure ulcers has been designed, including systematic biopsy of non healing ulcers
coupled with genetic analysis of ulcer borders.
At the Pio Albergo Tribuzio, (PAT) which is the largest Geriatric Institute in Milan, the prevalence of pressure
ulcers during years 2004-2008 varied from 9% to 14%, i.e. 110 out of 705 subjects (29F, 49M), whereas ulcers
of lower limbs were found in 35 (17M, 18F). In particular, in March 2009, in 473 subjects who were admitted
to the PAT-RSA, there were 42 subjects with pressure ulcers stage IV. There were 28 F and 14M. Diabetes
requiring treatment was found in 7 of 42 (insulin administration in 4 cases). Data from molecular biology were
found of particular help to guide complete removal of the border showing hyperexpression of c-myc and βcatenin, so facilitating early recovery using a multidisciplinary approach, that involves surgeons, diabetologists, dermatologists, orthopedicians, angiologists, geneticians and pathologists.
- 23 -
Oncology and Genetics Doctoral School
Medical Genetic Doctoral School
Medical Genetics
XXIV cycle
David Colecchia, BS
[email protected]
Tutor A. Renieri, M. Chiariello
Multiple primary malignancies: Yeta challange
We analyzed databases on 815 subjets who underwent surgery for colorectal cancer from 1985 to 2005 periodically followed up by clinical and instrumental examinations.
The aim of our follow up program is the detection of the first cancer representation and the prevention of
metachrone colic or extracolic malignancies.We detected MPM (Multiple Primary Malignancies)in 120 out of
815 patients (14.72%).Metachronous malignancies are more frequent than synchronous ones (75vs 45). The
mid time between two neoplasms is 40 months
Among metachronous neoplasms,extracolic ones are more frequent.In subjects that developed a colorectal
cancer after a first colorectal cancer, the last pancolonoscopy was performed 22.5 months before.Three
metachronous tumors found six mounths after first surgery have been considered misunderstood synchronous malignancies. The colic neoplasia localization, proximal or distal to the right flexure, is similar in patient
with unic colorectal cancer and in patient affected by MPM.
In 15 MPM patients suggestive criteria for hereditary colorectal cancer are present and these subjects have
been invited to genetical counselling and we are studing results.
Skin, breast and colonrectum are in this order the most frequent sites interested by multiple primary malignancies.Colonrectum is involved by MPM in a percentage from 30 % to 50 % according to Literature.
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Oncology and Genetics Doctoral School
Doctoral School of Oncology and Genetics
Oncological Genetics
XXIV cycle
Daniele Conti, MS
[email protected]
Tutor A. Giordano
Identification of the regulatory mechanisms of Cdk2/CyclinA inhibition by pRb2/p130
protein
Over the past decades, cancer research has been mainly aimed at identifying the molecular alterations
underlying cancer development, in order to design new drugs for targeted therapy.
Retinoblastoma (RB) family proteins pRb, p107 and pRb2/p130 are important cellular factors which play a
well-recognized role as tumor and growth suppressors. These proteins are actively involved in the negative
control of the cell cycle and their function is modulated via complex homeostatic processes, most of them
involving post-translational regulation of their phosphorylation status. Interestingly, the family members p107
and pRb2/p130 share the ability to physically interact and inhibit the kinase activity of the Cdk2/Cyclin A and
Cdk2/Cyclin E complexes. Regarding pRb2/p130, its inhibitory effect on Cdk2/Cyclin A activity has been attributed to the "spacer" region, in particular to a 39 aa-long pRb2/p130 spacer-derived peptide (Spa310, aa 641679) was selected as the sequence responsible for Cdk2/CyclinA inhibition.
We used a computational chemistry approach to select a pool of small molecules that mimic Spa310 activity.
The analysis of the CDK2-CyclinA crystal structure allowed us to select five hypothetical CDK2-CyclinA inhibitors from chemical libraries. We tested the antiproliferative effects of these five small molecules on cell lines
of different tumor types (lung and prostate cancer, osteosarcoma, mesothelioma and medulloblastoma) by the
MTS cytotoxicity assay. We observed a significant reduction in the growth rate of these tumor cells and we
focused our further analyses on the two most effective compounds. In order to rule out the potential cytotoxic effect on normal cells, we tested these molecules also on non-neoplastic cell lines. We found that they
have a significant minor effect on normal cells with respect to their tumoral counterpart. Preliminary FACS
analyses show that both the selected small molecules can induce apoptosis in lung cancer cell lines.
Additionally combinatory tests with cisplatin show that the use of our small molecules is able to decrease the
necessary concentration of the chemotherapic to reach the same cellular mortality in vitro.
To dissect the molecular mechanisms of these small-molecules-induced apoptosis we will also analyze by
western blotting and real-time qRT-PCR the expression of proteins involved in the regulation of cell cycle and
apoptosis. As a future objective, we intend to test these small molecules in mouse tumor xenografts in order
to evaluate their ability to inhibit tumor growth also in vivo.
This work is reported in:
Conti D et al. Small molecules mimicking the spa310 peptide from the spacer region of pRB2/p130 as potential anticancer
agents. Fifth Annual Scientific Conference – Istituto Toscano Tumori (ITT), Il Borro, San Giustino Valdarno (Arezzo), July 1,
2010.
This work is funded by:
Istituto Toscano Tumori (ITT), Human Health Foundation (HHF) and University of Siena.
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Oncological Genetics
XXV cycle
Martina Cozzi, MS
[email protected]
Tutor A. Giordano
New small molecule inhibitors of Src as potential candidates for cancer therapy
Accumulating data show that alterations in the activity of the tyrosine kinase Src play a key role in the development and progression of several human cancers. Src has been shown to be an important molecular target in cancer therapy.
This study aims at investigating the effects of new pyrimidine derivative Src inhibitors in a panel of tumors
that show a high Src kinase activity. Given the central role of Src in regulating several key processes in tumor
development we plan to analyze the effects of the Src inhibitors on these processes.
We have recently studied the effects of these molecules in medulloblastoma, the most common cerebellar
tumor of the central nervous system in childhood. Therapeutic approaches for medulloblastoma are currently
based on the combination of surgery, radiotherapy, and chemotherapy. Despite improvements in the overall
survival rate following the multimodality treatment, about one third of patients will have a recurrent disease
and current treatments cause neurocognitive sequelae. Therefore, there is a great need to develop new therapies that minimize adverse effects. Substantial progress has been made in understanding the molecular
mechanisms underlying medulloblastoma and in offering new targets for the development of more effective
and specific therapies. One possible target for medulloblastoma therapy is Src. A high Src activity was identified in medulloblastoma, suggesting that Src could have a key role in the development of this tumor. We
have examined the effects of the Src inhibitors in human medulloblastoma cells (Daoy and D283). We showed that the pyrimidine derivatives greatly reduce the growth rate of medulloblastoma cells compared with
a non-neoplastic nerve cell line (HT22). These compounds halt cells in the G2/M phase, and this effect likely
occurs through the regulation of cdc2 and CDC25C. Moreover, the exposure to pyrimidine derivatives induces apoptosis through modulation of the apoptotic proteins Bax and Bcl2, and inhibits tumor growth in a xenograft mouse model. Notably, the small molecules show major inhibitory effects on medulloblastoma cell
growth compared with the chemotherapeutic agents cisplatin and etoposide. In conclusion, our results suggest that Src ihibitors could be novel attractive candidates for the treatment of medulloblastoma or tumors
characterized by high Src activity.
This work is published in:
New pyrazolo-[3,4-d]-pyrimidine derivative Src kinase inhibitors lead to cell cycle arrest and tumor growth reduction of
human medulloblastoma cells.Rossi A, et al. FASEB J. 2010 Apr 29.
This work is funded by:
U.S. National Institutes of Health grants and by the Sbarro Health Research Organization (http://www.shro.org), the
Human Health Foundation Onlus Spoleto, Italy (http://www.hhfonlus.org), and the Teresa and Luigi de Beaumont Bonelli
Foundation (to A.G.).
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Colorectal and gastroesophageal diseases
XXIV cycle
Francesca Crucianelli, BS
[email protected]
Tutor G. Tanzini, M. Genuardi
Detection of constitutional epigenetic changes in multiple cancers by MS-MLPA
analysis
Aberrant methylation of CpG-islands has been shown to be associated with transcriptional inactivation of
tumor suppressor genes in a wide spectrum of human cancers. Recently, evidence has been accrued showing that constitutional epigenetic silencing abolishes gene expression.
This project focuses on the investigation of aberrant constitutional DNA methylation in mutation-negative
patients with multiple primary tumors.
The search has been performed on non cancerous tissues. In addition, we have analyzed tumor tissue, when
available.
Methylation analysis has been performed by a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay that allows detection of CpG methylation in 24 different tumor suppressor genes.
In a first phase, purification of DNA from formalin-fixed, paraffin-embedded (FFPE) tumor tissues has been
performed with QIAamp DNA Mini Kit followed by preamplification of total DNA. Subsequently, to improve
yield as well as DNA performance in MS-MLPA, the QIAamp DNA FFPE Tissue Kit has been used.
Constitutional methylation is then confirmed by methylation-sensitive high-resolution melting (MS-HRM)
and/or pyrosequencing.
So far, DNA samples from peripheral blood of 43 patients, have been investigated. For two of these, tumor
samples were available to compare methylation patterns.
No alteration of MS-MLPA peak ratios has been observed in this small subset.
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Medical Genetics
XXIII cycle
De Filippis Roberta, BS
Tutor A. Renieri
FOXG1 mutation leading to reduced chromatin affinity causes “Rett fruste” overlapping with EHMT1 phenotype
The Forkhead box G1 (FOXG1) gene, a brain specific transcriptional factor essential for the early development of telencephalon, located in 14q12, has been recently implicated in the congenital variant of Rett
Syndrome (RTT). Until now only 10 patients with FOXG1 point mutations have been reported with a quite
homogeneous phenotype including a severe neurological impairment in accordance with a clinical diagnosis
of congenital variant RTT. These patients do not show peculiar facial features in contrast with those with the
14q12 microdeletion syndrome. Here we report on two unrelated patients with a de novo FOXG1 point mutation, p.Gln46X and p.Tyr400X respectively, having a milder RTT phenotype according with RTT “forme fruste” and sharing strikingly similar facial features resembling the Kleefstra syndrome due to EHMT1 gene.
Although FoxG1 action as well as EHMT1 depends critically on its binding to chromatin, very little is known
about the dynamics of this process. Here we apply photobleaching strategies within the nucleus comparing
the wild type GFP tagged FoxG1 with the protein carrying pathological mutations. We report for the first time
that most of the FoxG1 fusion protein is transiently associated with chromatin in vivo and that mutations caused a mislocalization of FoxG1 and a dramatic alteration in chromatin affinity which is particularly high in the
two mutations reported here. Interestingly both FoxG1 and EHMT1 proteins interact with members of JARID1
family and are involved in modulation of the chromatin structure. In this perspective, the overlapping phenotype described in this paper could not be completely unexpected.
This work has been submitted to the Jurnal of Medical Genetics:
“FOXG1 mutation leading to reduced chromatin affinity causes “Rett fruste” overlapping with EHMT1 phenotype”
De Filippis R et al.
This work is reported in: “Mutazioni in FOXG1 che causano ridotta affinità per la cromatina, sono responsabili della sindrome di Rett nella variante “Rett fruste” con fenotipo sovrapponibile a quello da Mutazione in Ehmt 1” De Filippis et
al.SIGU 2010.
This work is founded by
PRIN 2008 to F.M. and by Telethon grants GTB07001 and GDP09117 to A. R..
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Medical Genetics
XXIV cycle
Vittoria Disciglio, BS
[email protected]
Tutor A. Renieri
Role of MDM2 T309G and TP53 R72P polymorphisms in modulation of variable phenotypic expression of retinoblastoma
Current evidence support the role of DNA repair and apoptosis gene polymorphisms as cancer modifiers. Two
common SNPs TP53 R72P and MDM2 SNP309 with known functional effects have been studied with contrasting findings in both sporadic cancer (gastric, lung, childhood ALL) and the inherited Lynch syndrome, and
in Li-Fraumeni syndrome a significant interaction between the germline TP53 mutation and the MDM2 SNP
has been shown. To investigate their role in hereditary retinoblastoma we genotyped the two SNPs by
Pyrosequencing® assays on blood DNA of 90 patients with known germinal RB1 mutation, 34 familiar. A
descriptive analysis showed an earlier age at diagnosis in patients with bilateral retinoblastoma than in those
with unilateral retinoblastoma (median age: 0.57 yrs vs 1.49 yrs, respectively, p<0.001). Since age of onset
is often nor exactly known, we considered bilaterality as a more robust measure of the variable genetic risk.
A multivariate logistic regression model adjusted for age and gender showed the risk of bilateral disease to
be: i) as for the type of RB1 mutation higher for splicing and missense mutations than for deletions, duplications, nonsense and frameshift mutations but not significantly so (OR=1.33; 95% CI 0.22 – 8.22); ii) as for the
MDM2 SNP309, significantly higher for the GG genotype than TT (OR=11.78, 95% CI 2.18 – 63.65) but not
significantly for TG; iii) as for the TP53 R72P SNP not significantly for the PP genotype. Our results suggest
for the first time that MDM2 may be modifier gene of Retinoblastoma as well.
This work is reported in:
Disciglio V. et al. MDM2 and TP53 are modifier genes of retinoblastoma. European Congress of Human
Genetics, Gothenburg, Sweden - June 12 - 15, 2010. Poster
This work is funded by:
FIRB grant to A.R., by the University of Siena grant PAR2006 to M. B., and by a grant on Retinoblastoma
from Istituto Toscano Tumori (ITT) to A.R.
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Colorectal and Gastroesophageal Disease
XXV cycle
Andrea Fontani, MD
Tutor G. Tanzini
Outcome of surgical trearment of colorectal cancer in the elderly
Aim of this study is to compare the clinical features and the perioperative and long term outcomes after primary surgery for colorectal cancer in the elderly population with those observed in younger patients.
All the patients over the age of 55 who underwent primary surgery for colorectal cancer in our clinic from 1988
to 2008 were included in this study and divided into two age groups: 55-75 years and >75 years considering
the age of diagnosis.
914 consecutive patients were enrolled in the study (352 >75 years). In the elderly group tumours were predominantly right sided and the overall number of comorbidities was statistical more frequent.
Elderly patients underwent emergency surgery more than the control group (p=0.0008). There were no significant differences between the two group in terms of curative and palliative resections.
The overall operative mortality rate was 5,9% in the Study group compared with 2,1% in the Control
Study(p=0,0033).
The overall 3-year, 5-year and 10-year survival rates were respectively 37%, 16,2% and 5,1% in the Study
group, compared with 52,3%, 35,1% and 24,7% in the Control group. (p=0,022, p=0,0001 and p=0,0001
respectively).
More patients were lost during the follow-up in the elderly group (p=0,0003) and more deaths unrelated to
cancer were found in the study group compared with the control group (p=0,0005).
The cancer specific mortality was similar between the two groups.
Elderly patients that underwent major colorectal resection have an acceptable perioperative morbidity, mortality and survival rate compared to younger patients.
Age alone should not be considered a reason to deny surgery to these patients.
This work is reported in:
Fontani A et al. Outcome of surgical treatment of colorectal cancer in the elderly. ESCP, Sorrento, 22-25
Settembre 2010.
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Oncological Genetics
XXIV cycle
Iris Maria Forte
[email protected]
Tutor A. Giordano
Gastric cancer and cell cycle regulation
Gastric cancer is one of the most diffuse neoplastic pathologies in the world whose environmental and
molecular causes, although deeply investigated, have not been completely clarified. Gastric cancer aetiology
is related to Helicobacter pylori infection, high intake of smoked and salted food and genetic alteration of Ecadherin gene, CDH1, a calcium-dependent cell.adhesion glycoprotein, the loss of which contributes to cancer progression by increasing proliferation, invasion and metastasis. Two histotypes of gastric cancer are
recognized: the diffuse histotype, characterized by poor differentiated secreting mucus cells, often associated to CDH1 germline mutation ; and the intestinal hystotype, characterized by a series of multi-defined steps
beginning with atrophic gastritis and finally developing into carcinoma and diffuse metastatis. Since molecular bases of gastric cancer are so far not clear, the aim of our work is trying to define the role of cell cycle
regulatory genes in the pathogenesis of the gastric cancer, focusing on the Rb family proteins. The pRb family
proteins (including pRb/p105, pRb2/p130 and p107) are involved in cell cycle regulation and their function
and/or expression is often lost in various kind of tumors. They also regulate growth processes and apoptosis above all by interacting with E2Fs transciption factors. Only few studies have been performed about the
role of Rb family proteins in gastric cancer. So far we have collected 14 tissue specimens of gastric cancer
with their corresponding normal gastric tissue and we studied the expression level of both protein and mRNA
of pRb2/p130 both by immunohistochemistry and by real-time PCR in all samples. Until now we found that
pRb2/p130 shows gene downregulation in all samples while the protein expression of pRb2/p130 does not.
We obtained the same results studying gene and protein expression of pRb2/p130 in 5 cell lines. However,
our statistical data do not show a direct correlation between pRb2/p130 gene level expression and grade of
tumor. A the present we are studying the expression of cyclin A2 in all tissues samples. In fact, Cyclin A2 is
a target of E2F4 transcription factor which is released after pRb2/p130 phosphorilation. So far we found that
Cyclin A2 show a gene upregulation, probably caused by hyperposphorilation or mutation of pRb2/p130. Our
future purpose are investigating on possible altered function of this protein and sequencing of its related gene
to detect possible mutation.
This topic is reviewed in:
Rb family proteins in gastric cancer Cito L., et al. Oncology reports (Accepted 05/05/2010)
This work is funded by:
Centro Ricerche Oncologiche Mercogliano, CROM.
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Medical Genetics
XXV cycle
Grillo Elisa, BS
[email protected]
Tutor A. Renieri
European Rett Database Network
Rett database network project started in 2008 in order to connect the already existing databases and to create a unified repository following an “adaptor approach”. In addition, this network allows data storage for users
who do not have a local computerized data management system. Presently, the network contains data of
1246 patients. The whole Italian (Italian Rett database and biobank), French (SYRENE), British (British Isles
Rett Syndrome Survey) and Spanish (Barcelona Rett database) cohort have being converted and patients
inserted from the pre-existing databases. In addition, patients from Israel, Denmark, Croatia and Serbia have
been inserted directly in the network web site. The access is now open worldwide to whom that want to join
(email contacts [email protected] and [email protected]). For those who do not have a pre-existing database there are two options: i) insert directly in the main archive (geographical and institution provenience will
be displayed); ii) construct a local or national archive connected with the main one. The web site is now
moved to the definitive address (https://www.rettdatabasenetwork.org) and it has been implemented with the
following 5 general pages (accessible by clicking on the top of the home page): i) "about this project"; ii) "how
to join"; iii) "access rules"; iv) "guidelines"; v) "consent form". During the next year we are planning to improve the database and to develop a data mining system, which can manipulate large scientific databases. This
international effort will be of great value in order to perform genotype-phenotype correlations, to study modifier genes, and to select subgroups of patients for clinical trials. The data are accessible to the participants
and to the scientific community according to rules that assure transparency and equity (published in the web
site).
This work is reported in:
A. Renieri et al. “Rett database network project”. IRSF’s 11th Annual Rett syndrome Symposium, Leesburg, Virginia,
U.S.A., June 27 - 29, 2010. Poster
This work is founded by: IRSF(International Rett Syndrome Foundation), E-RARE (European Network on Rett
Syndrome)
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Oncology and Genetics Doctoral School
Medical Genetics Doctoral School
Medical Genetics
XXII cycle
Valeria Guarnaccia, BS
[email protected]
Tutor A. Renieri, E. Garattini
Retinoic acid and breast cancer: how to improve the therapeutic effect on the basis
of molecular knowledge
Breast cancer is the second leading cause of cancer deaths in women. At least five distinct subtypes have
been described on the basis of gene expression profiling, with the most important determinants of these
subtypes being the presence or absence of the estrogen receptor alpha (ER) or the amplification/over expression of the Her2/ERBB2 locus. Breast carcinoma is a paradigmatic example of heterogeneity in the responses to retinoids. Clinical data indicate that only pre-menopausal women benefit from the use of the synthetic
retinoid, fenretidine, when the drug is used in an adjuvant setting. The major aim of the research program of
my PhD is to characterize the retinoid sensitivity/resistance in breast carcinoma and to define its molecular
mechanism across the ER alpha and Her2 pathway. Retinoids exert their pleiotropic effects by binding to specific nuclear receptors (RARs and RXRs) acting as nuclear transcription factors. Generally, breast carcinoma
cells expressing ER alpha are described as sensitive to, while the ER alpha-negative counterparts are refractory to the anti-proliferative activity of retinoids. On this basis, we have taken advantage of paradigms of ER
alpha-positive (MCF7) and ER alpha-negative (MDA-MB231) breast cancer cells to assess the role of ER
alpha signaling in determining ATRA sensitivity. After evaluation of the sensitivity of the cell lines and their stable transfectants restoring or silencing ER alpha, we performed gene expression and miRNA analysis for the
dissection of the molecular mechanisms underlying the cross-talk between ER alpha and the retinoid signaling pathways. Interestingly, we found that the mir-21 is selectively induced by ATRA in ER alpha-positive cells
but not in a panel of ER alpha-negative cell lines. This induction is the result of a transcriptional effect exerted by ATRA on the MIR21 gene. RNA silencing and over-expression experiments indicates that mir-21 could
counteract the anti-proliferative effect of ATRA possibly acting in a negative feed-back loop. As far as Her2 is
concerned, we considered that a significant fraction of Her2/Neu+ breast carcinomas are ER-, hence this type
of cancer is predicted to be refractory to retinoids. Her2/Neu+ is often the result of an amplification of the corresponding gene (ERBB2) which maps to chr17, 650 kb upstream of RARα (RARA locus). For this reason
we evaluated whether a proportion of Her2/Neu+ and ER- breast cancer cells are characterized by co-amplification of ERBB2 and RARA, and hopefully could represent a new therapeutic target for retinoids. Using real
time PCR on genomic DNA from 76 patients positive for Her2 amplification, we found that at least 20% harbours RARA amplification. To evaluate whether RARA co-amplification and consequent RARα over-expression was associated with retinoid sensitivity, we turned to a panel of representative breast carcinoma cell
lines. We found that Her2-RARalpha co-amplified cell lines are particularly sensitive to retinoic acid. Moreover
we found in SKBR3 cells that ATRA strongly synergize with lapatinib, a small tyrosine kinase inhibitor, used
in the treatment of Her2 amplified breast carcinomas thus opening the doors for a novel therapeutic
approach.
Part of this work has been submitted to Oncogene. Terao et al. Induction of miR-21 by retinoic acid in estrogen-receptorpositive breast carcinoma cells: biological correlates and molecular targets.
Part of this work is reported in: Garattini et al. “Combinations of retinoids and lapatinib in the treatment of Her2/Neu-positive breast carcinomas with co-amplification of the ERBB2 and RARA genes”. AACR 101st ANNUAL MEETING 2010, April
17-21,2010, Washington.
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Hepatobiliopancreatic Disease and Multitumoral syndromes
XXV cycle
Valentina Guercio, BS
[email protected]
Tutor F. Cetta
PM chemical characterization and differences in cytotoxicity versus pro-inflammatory potency of different PM fractions in human epithelial lung cells
Air pollution in Milan causes health concern due to the high concentrations of particulate matter (PM10 and
PM2.5). The aim of my study was to investigate possible seasonal differences in PM10 and PM2.5 chemical
composition and their biological effects. The PM was sampled during winter and summer seasons. The winter PMs had higher levels of PAHs than the summer samples which contained a greater amount of mineral
dust elements. The PM toxicity was tested in the human pulmonary epithelial cell lines BEAS-2B and A549.
It was found that, whereas A549 cell viability was not significantly reduced after summer and winter PM exposure, summer PM had no significant effects on BEAS-2B viability, whereas winter PM treatment induced a
decrease in cell viability. In addition, whereas both winter and summer PM2.5 produced only a slight increase in IL-8 release, winter PM10 induced a 5-fold increase in IL-8 release in treated cells, and summer PM10
induced a 20-fold increase (p<0,05) in IL-8 expression. In particular, BEAS-2B resulted more responsive to
PM treatment than A549. Winter PMs were more cytotoxic than summer PMs; Summer PM10 had a higher
proinflammatory potential, which could be partly due to biological components (LPS). These results underline that the in vitro responsiveness to PM may be cell line dependent and suggest that the PM different properties may trigger different endpoints such as inflammation, perturbation of cell cycle and cell death.
This work is reported in:
Cetta, F. et al. A comparative study on the outcomes between in vitro results of PM incubation with cell-lines and clinical
effects in children. The Milan Prolife Project. 1048 “International Aerosol Conference (IAC 2010) Helsinki, Finland 29
August – 3 September 2010.
This work is funded by:
CARIPLO Grant to POLARIS and by the PROLIFE-Project, City of Milan, Italy.
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Oncological Genetics
XXII cycle
Baharak Khadang, MD
[email protected]
Tutor A. Giordano
New Strategies for the Study and Treatment of Malignant Mesothelioma
Malignant mesothelioma (MM) is a rare highly aggressive tumor whose principal risk factor is exposure to
asbetos. The extensive use of asbestos during the twentieth century, together with the long-latency time (3040 years after the first exposure), determined an increase in incidence, which is expected to reach a peak
over the period 2020-2050. The prognosis is extremely poor, with a median survival between 10 and 17
months from clinical presentation. At present there is no known curative modality for MM. Understanding the
biological mechanisms underlying mesothelioma development, progression and resistance to therapy is
essential to identify new targets for the development of more effective and specific therapies and to identify
new diagnostic, prognostic and predictive markers. Given its possible role in MM pathogenesis, one potential
therapeutic target could be the Src kinase. We recently showed that new pyrimidine derivative Src inhibitors,
which bind the ATP pocket of the Src kinase, have antiproliferative and proapoptotic properties in several
tumor types. We evaluated the effect of these new Src inhibitors on a panel of human MM cell lines expressing the active form of Src (phospho-Src Y419) and also on a normal mesothelial cell line not expressing the
active form of Src. Our results showed that these Src inhibitors exert a significant proapoptotic effect on MM
cell lines, without affecting the normal mesothelial cells. Interestingly, we found that Src inhibition leads to
nuclear stabilization of the cyclin-dependent kinase inhibitor p27. This finding is remarkable considering that
loss of nuclear p27 expression is a well-established adverse prognostic factor in MM. Therefore, our observations provide a new rationale for the use of Src inhibitors in MM therapy.
Because miRNAs, a new family of gene expression regulators, have a crucial role in cancer development,
we analysed the miRNA expression signature in patients with MM compared to normal pleural samples in
order to identify new possible diagnostic, prognostic and predictive markers. We identified several miRNAs
that are downregulated or upregulated in MM tumor samples compared to normal pleura. So far we have confirmed by real-time qRT-PCR the differences observed by microarray for 10% of the miRNAs that resulted
more significantly deregulated in tumors. We focused our further analysis on miR-145, because it has proved
to be a putative tumor-suppressive miRNA in several tumor types and it is markedly downregulated in the
patient’s group with respect to normal controls. We observed a significant downregulation of miR-145 in
biphasic and sarcomatoid samples with respect to epithelioid specimens indicating that loss of miR-145
expression is a feature of the more aggressive tumor types. Consistently, we also observed a correlation between low miR-145 expression and a high proliferation index, measured by Ki67 immunohistochemical analysis. Finally, ROC curve analysis demonstrated that miR-145 could be a good prognostic and diagnostic marker for malignant mesothelioma.
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Oncological Genetics
XXIII cycle
Raffaele La Montagna, MS
[email protected]
Tutor A. Renieri
Androgen receptor and PIN1 in prostate cancer
Prostatic adenocarcinoma is the most frequently diagnosed malignancy and second leading cause of cancer
death amongst men in the United States. Among different players, many lines of evidence indicate that the
androgen receptor (AR) functions as a positive regulator of cell proliferation in Prostate Cancer, and androgen deprivation therapy is still the standard treatment for metastatic disease. AR is a member of the steroid
hormone receptor subfamily of ligand-regulated nuclear receptors, and its natural ligands are testosterone
and 5α-dihydrotestosterone (DHT). As other steroid receptors, AR is a modular protein that contains an N-terminal transactivation domain, a conserved DNA-binding domain (DBD), and a C-terminal ligand-binding
domain (LBD). Mechanistic investigation has revealed that AR acts as a master regulator of G1-S phase progression, regulating the activity of G1 cyclin-dependent kinase (CDK) proteins and induce
phosphorylation/inactivation of the retinoblastoma tumor suppressor (RB).
Many evidences suggest that Pin1 regulate the activity of AR. Pin1 is an isomerase specific of pSer/Thr-Pro
motifs that catalyzed the conformational switch from cis to trans, which is especially important because Prodirected kinases and phosphatases are conformation-specific and act only on the trans conformation.
Androgen receptor interacts with β-catenin and can suppress its coactivation of T cell factor 4 (Tcf4) in prostate cancer cells. Pin1 abrogated the ability of AR to antagonize β-catenin/Tcf4 binding and transcriptional
activity. Abrogation of this interaction can enhance β-catenin/Tcf4 signaling and contribute to aggressive biological behavior in Prostate Cancer. In addition, higher Pin1 expression in primary Prostate Cancer cells is
correlated with disease recurrence, and Pin1 expression was found markedly increased in metastatic
Prostate Cancer. Our preliminary data showed that Pin1 can form a protein complex with AR. We are currently investigating which domains are involved in the Pin1/AR interaction. We are modulating the expression
of Pin1 by shRNA and ectopic expression to understand how the AR activity is affected.
Fig. 1: AR-cell cycle crosstalk
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Hepatobiliopancreatic Disease and Multitumoral Syndromes
XXIII cycle
Laviano Paolo, MD
[email protected]
Tutor F. Cetta
Study of obesity prevalence, body mass index including energy consumption measured by accelerometer and pollution related health effects in children attending primary school in Milan
Traffic related pollution adversely affects lung function development. In addition, obesity and reduction of
physical activity have been suggested as possible co-factors, determining the occurrence of functional alterations, or early changes in individual susceptibility to the “pollution diseases”, which can be responsible for
cardiovascular diseases later in life.
228 children (±8 years) were enrolled from 2 primary schools in different sites of Milan with a different traffic
related exposure: School1 near a large park, School2 close to main crossroads. Daily levels of PM10 and
PM2,5 were measured both outdoor and indoor, during two 2-week different campaigns. Children underwent
skin prick testing for inhaled allergens, analysis of Fractional Exhaled Nitric Oxide (FeNO) and spirometry.
In particular, in 188 children (62M, 126F), in addition to anthropometric data concerning parents,
a questionnaire concerning dietary habits and timing and numbers of meals, physical activity of children was
analyzed and quantitatively measured using a portable accelerometer (Lifecorder PLUS, KENZ), to be used
for at least 3 days (2 working days +1 week-end) for at least 8 hours for day (N= 180).
30 children (16%) made a regular physical activity for less than 30 minutes; 127 (67,6%) between 30 and 60
minutes, whereas 21 (11,2%) for more than 1 hour. The percentage of children with FeNO values <5ppb in
School1 was higher (almost double) than in School2 (p=0,02). In 73% of children attending the School2
FeNO was between 5 and 20ppb. The percentage of asthma exacerbations in the previous 12 months was
higher in children from School2 (p=0.05). On the contrary, the prevalence of persistent allergic rhinitis in children allergic to grass pollen was higher in School1 (p=0.03). In particular, the latter children also had a greater activity limitation, due to rhinitis and concomitant conjunctivitis (p=0.03).
Continuous on field monitoring of the various types of PM and analysis of clinical outcomes and hospital
admission shows that: -Short durations PM10 peaks, even reaching high concentrations (up to 1000 µg/m3)
have no evident consequences on children health and are usually induced by trivial causes (resuspension of
crustal components).- There is an enormous daily variability in PM10 concentration, and a wide range of clinical outcomes, showing seasonal variability, likely related to different seasonal composition of PM.
Traffic could be responsible at least in part for the different air quality, but individual susceptibility and seasonal changes are also major determinants of clinical outcomes. Preliminary data concerning concomitant
obesity and overweight, because of the too short observation period, resulted insufficient to detect a facilitating role of overweight, dietary habits and reduction of physical activity, in the occurrence of more severe clinical outcomes.
This work was supported by the PROLIFE Project, City of Milan, Italy
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Medical Genetics
XXV cycle
Gabriella Livide, BS
[email protected]
Tutor A. Renieri
Mutational screening in the RB1 gene
The RB1 gene (Gene Bank accession number L11910) consists of 27 exons and of a promoter region containing binding motifs for transcription factors such as Sp1, ATF and E2F. It encodes for a 928 amino acids
phosphoprotein that is part of a small family of nuclear proteins, acting on cell cycle regulation. The pRB pathway plays an important role in controlling proliferation and is frequently perturbed in human tumors.
Retinoblastoma is a childhood tumor of the developing retina, occurring either in a sporadic or in a hereditary
form. Sporadic tumors are unilateral, whereas hereditary tumors are often bilateral and multifocal; the latter
show an earlier age of onset, and are transmitted in a dominant mode with a penetrance of about 90%. The
first model to explain tumor development was proposed by Alfred Knudson in 1971; according to this model,
two mutational events (M1–M2) in RB1 gene represent the first rate-limiting step in tumor development. Next
high resolution studies demonstrated that these mutational events are followed by further genomic rearrangements (M3-Mn) that involve oncogene/oncosuppressor, necessary for malignant transformation of retinoblastoma. The aim of my study was to detect the loss/alteration of pRB function, through the application of
MLPA, DHPLC and Automatic Sequencing techniques. I studied a cohort of 27 patients: 13 unilateral case
(12 sporadic, 1 familial) and 14 bilateral cases (12 sporadic, 2 familial). Among bilateral cases I found 13
mutations: 8 non sense mutations, 3 splice mutations, a deletion involving exon 20, and a partial gene deletion involving exons 1-18; among unilateral cases we found a nucleotide substitution involving the promoter
and a non sense mutation. The consequence of the nucleotide substitution involving the promoter region is
that the nuclear factors do not bind to the mutant sequences; these nuclear factors are necessary for the
expression of the Rb gene and the suppression of cancer.
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Medical Genetics
XXIV cycle
Bruno Lorenzi, MD
[email protected]
Tutor F. Cetta
Evaluation of anorectal function after radiotherapy in patients treated for rectal cancer
Radiotherapy improves local control in rectal cancer treatment and it is currently recommended in an increasing number of patients. However, there are few reports on the influence of radiotherapy on anorectal function.
In this study, we first investigated the functional changes of the internal anal sphincter (IAS) following radiotherapy in vitro. We collected IAS strips from patients undergoing abdominoperineal resection or proctectomy
and monitored the responses to electrical field stimulation (EFS) and different drugs. Five patients were treated by surgery alone, and six received pre-operative radiotherapy. There were significant differences in the
responses of control and irradiated strips to EFS (p<0.01), Nω-nitro-L-arginine (p<0.01), carbachol (p<0.05)
and phenylephrine (p<0.05).
Then, we evaluated the anorectal function and clinical outcome of patients undergoing anterior resection for
rectal cancer who received preoperative radiotherapy, and compared with the results of patients treated with
surgery alone. Twenty-one patients had received preoperative radiotherapy and 13 patients had been treated
with surgery alone.
Anorectal function was evaluated using questionnaires, anorectal manometry and endoanal ultrasound.
Functional outcome was disappointing in both groups comparing with preoperative results. In particular, rectal compliance was significantly reduced after surgery. However, irradiated patients had significantly more
symptoms of faecal incontinence (57 vs. 26 percent, p<0.01), soiling (38 vs. 16 percent, p<0.05) and bowel
movements (20 vs. 10, p<0.05) compared to controls. At anorectal manometry, irradiated patients had significantly lower resting (35 mmHg vs. 62 mmHg, p<0.01) and squeeze pressures (104 mmHg vs. 143 mmHg,
p<0.05), and showed more scarring of the anal sphincters at endoanal ultrasound (33 vs. 13 percent, p<0.05).
In conclusion, anterior resection alone adversely affects the functional outcome of patients treated for rectal
cancer, resulting in increased evacuation disorders and symptoms of faecal incontinence. In particular, anorectal function after rectal surgery with or without radiotherapy is hampered because of a decreased rectal
compliance. Radiotherapy further worsens anal continence mainly by impairing IAS function.
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Hepatobiliopancreatic Disease and Multitumoral Syndromes
XXI cycle
Giuliana Malagnino, PCS
[email protected]
Tutor F. Cetta
Intrinsic toxicity, inflammatory potency and individual susceptibility in the occurrence
of health damage from Particulate Material (PM)
Reactive oxygen species (ROS)—which are considered one of the main cellular stressors generated by PM
exposure—may produce genomic hypomethylation and increased expres¬sion and activity of inducible nitric
oxide synthase (iNOS) not only in vitro, but in humans exposed to particulate matter (PM). Although this finding
is expected, it is a step forward, based on DNA adduct generation produced by polycyclic aromatic hydrocarbons (PAHs) and other PM compo¬nents, namely transition metals. Alterations of DNA methylation of the promoter is a common finding in environmental-related chronic or cancerous diseases.
The occurrence of DNA adducts or mutations in some cells or tissues due to exposure to PAHs or diesel exhaust
does not necessarily induce clinically evi¬dent outcomes in the future, because each individual is endowed with
a wide variety of natural defenses and repair mechanisms that usually overcome every type of DNA damage.
Chronic exposure to toxic or carcinogenic environmental substances does not elicit the same results in all individuals. The final clinical outcome (cancer, pulmonary fibrosis) seems to be less dependent on the toxic potency
of the pollutant or of the exposure dose and more on the individ¬ual susceptibility of the host. This approach
should facilitate a better understanding of the < 5% incidence of mesotheliomas in subjects with the same chronic exposure to asbestos, or the absence of health effects in husbands with chronic occupational exposure to
asbestos but the occurrence of mesotheliomas in wives with minor indirect exposure from their husbands.
The final clinical outcome (e.g. cancer) does not depend on the first DNA adduct formed or a genetic mutation
produced by xenobiotics but is greatly influenced by individual susceptibility or resistance.
- 40 -
Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Medical Genetics
XXII cycle
Elena Marcocci, BS
[email protected]
Tutor A. Renieri
Autosomal Alport syndrome: a new model including both dominant and recessive
inheritance
Alport syndrome is a clinically and genetically heterogeneous nephropathy characterized by irregular thinning,
thickening, and splitting of the glomerular basement membrane often associated with hearing loss and ocular
symptoms. The majority of cases, about 85%, are transmitted as an X-linked form due to COL4A5 mutations.
Less than 15% of cases are autosomal recessive and dominant forms due to mutations in either COL4A3 or
COL4A4 genes. My research project focus on the analysis of the COL4A4 and COL4A3 genes in a large cohort
of patients by DHPLC (Denaturing High Performance Liquid Chromatography) and automated sequencing.
Molecular analysis revealed in COL4A4 or COL4A3 gene 24 different mutations: 18 autosomal dominant forms
(from 20 different families) and 6 autosomal recessive forms (from 4 different families). The single base deletion
p.P629fsX652 was found in two different unrelated families coming from Marsala, while the missense mutation
p.G1045V was found in three different unrelated families coming from Castelfranco Veneto. This could lead us
to think of a possible founder effect for this two mutations. In one of the three families with p.G1045V mutation,
an initial clinical analysis of the proband, of the brother and the sister showed that they developed renal disease at the age of 31, 26 and 19 years respectively, suggesting the hypothesis of a recessive Alport syndrome.
This hypothesis is confirmed by the fact that the proband’s father present a mutation with a normal clinical profile. However, the older brother, aged 44, showed only microscopic hematuria and proteinuria suggesting an
autosomal dominant form. This suggest a new model including both dominant and recessive inheritance. In conclusion, it is very difficult to predict the prognosis in a patient with an heterozygous mutation in either the COL4A3
or the COL4A4 gene. A correct diagnosis and prognosis is based on a combination of a comprehensive clinical
investigation of all family members, including examination of renal and extra-renal signs of Alport syndrome in
older members, associated with a broadly formal genetic analysis of the pedigree.
Part of this work is published in:
Marcocci et al. Autosomal Dominant Alport syndrome: molecular analysis of the COL4A4 gene and clinical outcome.
Nephrol Dial Transplant. 2009 May; 24(5):1464-71.
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Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Oncological Genetics
XXIV cycle
Monica Mischitelli, BS
Tutor A. Giordano, V.A. Pietropaolo
Prostate cancer: the influence of STAT3 and the presence of Human Polyomavirus BK
in cells
Prostate cancer (PC) is a common cause of death and is incurable in the androgen-refractory phase. Multiple
factors contribute to PC development, especially, signal messengers and transcription factors such as the Signal
Transducer and Activator of Transcription 3 (STAT3). STAT3 is abnormal activated in PC cells where it changes
gene expression leading to transformation.
Oncogenic infectious agents could also play a role in PC. The Human Polyomavirus BK (BKV) could be a candidate since it is an ureteliotropic virus, almost 90% of adults are seroconvert and the in vitro oncogenesis is proven. In fact, BKV large T-Antigen (TAg) could deregulate cell’s cycle by sequestering tumor suppressors as p53.
In this study, fresh biopsies of 15 patients (median age of 60) with clinically pT3a androgen-refractory PC, were
analysed. STAT3 target genes, in PC gene profile, were detected using ONCOMINE database and their expression was investigated using RetroTranscriptional Quantitive PCR (RT-QPCR). BKV DNA and AgT RNA were
searched using Quantitative PCR (QPCR) and RT-QPCR. As controls for BKV, non tumour biopsies of the same
patients were analyzed. Results showed that STAT3 target oncogenes such as c-myc, cdc25A, survivin and
Erp57 was overexpressed, BKV DNA was found in 3/15 patients and TAg mRNA was not detected. About controls, BKV was detected only in one. Data underline that STAT3 has a causal role in oncogenesis and is a target for cancer drug discovery and therapies. About BKV, DNA presence doesn’t exclude viral pressure on cell
transformation. Nevertheless, more investigations are required to elucidate how STAT3 influence gene expression, how cytokines could influence STAT3 response and finally if really BKV could operate on PC susceptibility.
- 42 -
Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Medical Genetics
XXIV cycle
Mafalda Mucciolo, BS
[email protected]
Tutor A. Renieri
Copy Number Variations analysis in Autism Spectrum Disorders
Autism Spectrum Disorders (ASDs) have a complex and heterogeneous aetiology with a strong evidence of a
genetic involvement. To assess the frequency and type of copy number variations (CNVs) in ASD, a cohort of
95 patients has been selected and analyzed by oligo array-CGH with a functional resolution of nearly 100 kb.
Array-CGH resulted negative in 60 patients while in 35 at least one rearrangement was identified. A total of 49
rearrangements was identified: 22 deletions and 27 duplications. Among the 35 patients with CNVs the M:F ratio
is higher than expected resulting in 6:1. Seven CNVs turned to be pathogenic: two de novo (del16p11.2; del
Xq12) and 5 located in known autism susceptibility regions (dup15q13.3, dup16p13.1, del15q11.2, del11p12,
dup17q12). The 7 patients with pathogenic CNVs were all males with intellectual disability (ID). The majority presented with congenital anomalies (MCA) and dysmorphisms (57.1%); none suffered from epilepsy. Among the
cohort of 60 patients without CNVs the M:F ratio resulted in 5.6:1, as currently reported in ASDs. ID was present
in 96.5%; MCA and dysmorphisms were present respectively in 21% and 63.2%. Epilepsy rate was 19.3%. The
detection rate of CNVs in our series of patients is 7.4%. Patients with pathogenic CNVs differed from the cohort
without any CNVs for the presence of congenital anomalies, more frequent in the first group. Furthermore, the
rate of epilepsy found in the all group was 19.3%, while in the subgroup of patients with pathogenic CNVs (n=7)
epilepsy was not detected. Finally, three patients presented two inherited rearrangements each, one inherited
by the mother and the other inherited by the father, leading to hypothesize the possibility of a digenic or multigenic inheritance.
Part of this work is reported in:
Mucciolo et al. Autism Spectrum Disorders and Copy Number Variations. 6th International DECIPHER Symposium, Hinxton
(UK), May 19-21, 2010. Oral presentation
Mari et al. Array-CGH analysis in Autism Spectrum Disorders. 6th International meeting on cryptic chromosomal rearrangements and genes in mental retardation and autism, Troina, April 22-24, 2010. Oral presentation
Mucciolo et al. Autism Spectrum Disorders: emerging data from Copy Number Variations analysis. European Congress of
Human Genetics, Goteborg, June 14-16, 2010. Poster
Canitano et al. CNVs in Autism Spectrum Disorders. American Academy of Child & Adolescent Psychiatry, New York City, NY,
October 27-31, 2010. Poster
- 43 -
Oncology and Genetics Doctoral School
Oncology And Genetics Doctoral School
Oncological Genetics
XXV cycle
Olayinka Olabinjo, BS
[email protected]
Tutor A. Giordano
Bioinformatics Approach to The pRb pathway in cancer initiation and progression
pRb is the key gene in a rare pediatric eye neoplasm (sporadic and hereditary) arising from retinal cells that
harbor either a deletion or mutation inactivation of both pRb alleles. pRb is consider a bona fide tumor suppressor gene. Its mutation and deletion is shared by several other malignancies. In a succinct word, pRb is
considered one of the hallmarks of human malignancies. pRb prevents the cell from replicating damaged
DNA by preventing its progression along the cell cycle through G1 (first gap phase) into S (synthesis phase).
pRb binds and inhibits transcription factors of the E2F family, which are composed of dimers of an E2F protein and a DP protein. The transcription activating complexes of E2 promoter-binding–protein-dimerization
partners (E2F-DP) can allow the cell into S phase. As long as E2F-DP is inactivated, the cell remains stalled
in the G1 phase. When pRb is bound to E2F, the complex acts as a growth suppressor and prevents progression through the cell cycle. The pRb-E2F/DP complex also interacts with histone deacetylase (HDAC)
protein to the chromatin, reducing transcription of S phase promoting factors, further suppressing DNA
synthesis
We are interested in finding pRb pathway in relation to other key players using bioinformatics approach.
Deregulations and mutations in this pathway are observed in most human cancer and the data analysis of
this pathway is still on the developmental stage and more complex structural analysis of the pRb pathway will
be undertaken. On the final analysis, a more thorough experimentation using CellDesigner software that will
be translated into BioPAX format will be performed and the result will be tested with the existing pathway databases. The construction of this pathway will serve among other purposes, to provide a map of pRb and other
related proteins that not only serve as a reference and a tool to understand the pathway but we will be able
to predict its behavior in response to different types of deregulations. As a long-term goal, we will connect the
pRb pathway with other pathways involved in the tumorigenesis with the scope to design new specific molecules for a multi-drug approach.
Diagram: Compreshensive cyclins and cell cycle regulation
showing RB pathways - courtesy of SABiosciences.
This work is funded by Sbarro Health Research Organization.
- 44 -
Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Oncological Genetics
XXIV cycle
Marco Pacifici
[email protected]
Tutor A. Giordano, F. Peruzzi
Detection and role of mir146a in HIV-clinical samples
MicroRNA-mediated regulation of gene expression appears to be involved in a variety of cellular processes,
including development, differentiation, proliferation and apoptosis.
Interestingly, miRNAs are secreted actively as free miRNAs or through microvesicles (MVs), suggesting that
these miRNAs may function outside the cells in which they are produced.
Mir146a is thought to be involved in the regulation of the innate immune response, and its expression is increased in tissues associated with chronic inflammation.
In our previuos study, we have analyzed changes in the expression of mir146a in primary human fetal microglial
cells upon infection with HIV and we have found increased expression of mir146a.
Furthermore, we have showed that CCL8/MCP-2, a ligand for the CCR5 chemokine receptor and a potent inhibitor of CD4/CCR5-mediated HIV-1 entry and replication, is a target for mir146a in HIV-1 infected microglial
cells.
Accordingly, overexpression of mir146a prevented HIV-induced secretion of MCP-2 chemokine, suggesting a
role for mir146a in the maintenance of HIV-mediated chronic inflammation of the brain.
Presently, we are focusing on the role of free and MVs-released mir-146a in the settings of HIV, in particular on
the capability of mir146a to be uptaken by myeloid dendritic cells (DC) and, eventually, on the modulation of different cellular events, including the innate immune response.
This work is published in:
CCL8/MCP-2 is a target for mir-146a in HIV-1-infected human microglial cells. Rom S. et al. Faseb J. 24, 000-000 (2010)
This work is funded by:
Francesca Peruzzi NIH Grants
- 45 -
Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Medical Genetics
XXII cycle
Filomena Tiziana Papa, MS
[email protected]
Tutor A. Renieri
Diagnosis process assessment of Array-CGH analysis
The use array-CGH (comparative genomic hybridization) has had a great impact on the practice of medical
genetics. This technology has been used to expand the phenotypes of well recognized clinical entities, determine the genomic lesions in known conditions, discover new syndromes, assess the prevalence of mosaicism, and ascertain the unexpected frequency of copy number variants (CNVs) across the genome. We performed oligo array-CGH analysis in DNA extracted from 330 patients with mild to severe mental retardation
associated with facial dysmorphisms and/or congenital anomalies. We identified 61 CNVs, ranging from 0.06
to 2.9 Mb, that were not previously described in the Database of Genomic Variants. In all cases, the same
rearrangement was inherited from a healthy parent. Fifty nine of the rearrangements were private, while
del17q12 (Fig. 1) was found in 3 patients and a del9p24.1 (Fig. 2) was found in 2 patients. Fourteen of the
rearranged regions contain disease genes. It is important to note that the presence of CNVs may be of difficult interpretation for the geneticist since some CNV may comprise benign genomic variants along with pathogenic rearrangements. Furthermore, concurrent variations in the other alleles or in another chromosome may
influence the phenotype. In order to firmly establish the clinical phenotype associated with a rearrangement,
it is of critical importance to examine additional patients with the same rearrangements, preferably through
collaborative efforts among several genetics units, and to update frequently the database and literature.
This work is published in:
Mencarelli MA et al. Private inherited microdeletion/microduplications: implications in clinical practice.
Eur J Med Genet. 2008 Sep-Oct;51(5):409-16.
This work is funded by:
Progetto ordinario-Ministero della Salute: Genomic structural variation studies in mentally retarded and normal individuals in
Italy (2008-2010).
- 46 -
Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Medical Genetics
XXIII cycle
Veronica Parri, BS
[email protected]
Tutor A. Renieri
Identification and characterization of deletions and duplications by MLPA in patients
with Cohen syndrome
Cohen syndrome is a rare, clinically variable autosomal recessive disorder associated to mutations in COH1
gene and characterized by mental retardation, postnatal microcephaly, facial dysmorphisms, ocular abnormalities and intermittent neutropenia. About 100 mutations of COH1 have been identified so far, most of
which are point mutations identified by automatic sequencing. Recently, MLPA was used to screen for COH1
large rearrangements instead of quantitative PCR, that is prone to miss a high fraction of mutated alleles. In
14 patients MLPA allowed us to disclose 11 deleted and 4 duplicated COH1 alleles. Three Italian families shared the same deletion, spanning exons 6 to 16, already reported in a large Greek consanguineous family.
Haplotype analysis suggested that the recurrent deletion is due to an ancestral founder effect in the
Mediterranean area. Two other deletions, spanning exons 4–16 and 40–43, have been already reported in
the Northern European population. In this case, these could be independent mutations favored by the presence of repeated elements located at the breakpoints. MLPA also identified three duplications spanning
exons 4–13, 20–30 and 57–60. Until now, COH1 intragenic duplications have never been reported in Cohen
syndrome. These rearrangements probably led to a frameshift and a premature truncation of the protein, as
demonstrated by sequencing the breakpoints of one of them. In conclusion, our study confirms that COH1
copy number variations are a frequent cause of Cohen syndrome and consist of intragenic deletions as well
as duplications. Therefore, the use of MLPA for molecular analysis of COH1 gene is mandatory in the molecular diagnosis of Cohen syndrome.
This work is published in:
Parri V. et al. High frequency of COH1 intragenic deletions and duplications detected by MLPA in
patients with Cohen syndrome. Eur J Hum Genet. 2010 May 12.
- 47 -
Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Oncological Genetics
XXII cycle
Flavio Rizzolio, MS
[email protected]
Tutor A. Giordano
Pin1 controls cell cycle progression through interaction with pRB
Normal cells became tumor cells through deregulation of multiple pathways. Much evidence suggests that
each type of tumors involves different proteins so that each type of cancer cells is different from the others.
Nevertheless, there are some pathways that are altered in many tumors and RB pathway is one of the most
important. pRB controls the cell cycle through the interaction with E2F transcription factors. These interactions are regulated during cell cycle by a phosphorylation mechanism. Ser or Thr followed by Pro are major
phosphorylation motifs in the cells but their significance was obscure until the discovery of the PIN1 protein
(protein interacting with NIMA (never in mitosis A)-1).
Pin1 is an isomerase specific of pSer/Thr-Pro motifs that catalyzed the conformational switch from cis to
trans, which is especially important because Pro-directed kinases and phosphatases are conformation-specific and act only on the trans conformation. In vivo and in vitro data have demonstrated that Pin1 is involved
in many aspects of cell cycle control. PIN1 was originally identified and defined as a protein that function in
mitosis. Since than, a plethora of protein targets have now been discovered many of which are involved in
the G0, G1/S control. We have demonstrated that Pin1 control cell proliferation through direct interaction with
pRB protein. The interaction is phosphorylation-dependent and it is also necessary for pRB phosphorylation.
An increase number of cells in G1 is observed in Pin1 knockdown cells. No apoptosis or senescence is detected. These results suggest that Pin1 can function as oncogene and Pin1 represents a excellent “druggable”
target since it appear a not essential gene in normal cells.
- 48 -
Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Medical Genetics
XIII cycle
Dalila Rondinella, BS
[email protected]
Tutor A. Renieri
QF-PCR as a tool for rapid prenatal diagnosis
Rapid prenatal diagnosis of major chromosome abnormalities can be performed using Short Tandem Repeats
(STRs) amplified by the Quantitative Fluorescence-Polymerase Chain Reaction (QF-PCR).
The Aneufast QF-PCR Kit allows the detection of aneuploidies involving chromosomes 13, 18, 21, X and Y
with 100% sensitivity and specificity, reporting results in 24-72h. This kit contains a total of 29 markers in six
mixes ready to use that amplify selected STRs and the gender determining sequences Amelogenin-SRY.
From May 2008 until today, 976 prenatal samples arrived and we have analysed by QF-PCR 386 samples:
232 amniotic fluids and 148 chorionic villus. The most common indications for prenatal diagnosis in our cohort
were advanced maternal age (86,4%), ecographic anomalies (5,5%), and biochemical risk (8,1%). QF-PCR
assays detected two cases of trisomy 13, five cases of trisomy 18, nine cases of trisomy 21 and one case of
triploidy which were confirmed by traditional karyotyping.
We have also a small casistic of abort samples: 23 cases analysed by QF-PCR that detected six cases of trisomy 21 and a case of X monosomy.
Our results are in accordance with recent studies, that following the analysis of several thousand samples,
have shown that this rapid approach has a very high rate of success and it could reduce the need for cytogenetic investigations. The main advantages of the QF-PCR are its accuracy, speed, automation, and low
cost that allows very large number of samples to be analyzed by few operators. For these reasons, in a near
future, this kind of assay could replace cytogenetic analyses.
- 49 -
Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral school
Medical Genetics
XXII cycle
Ariele Spanhol Rosseto, BS
[email protected]
Tutor A. Renieri
Molecular work-up of patients with the congenital variant of Rett syndrome: analysis
of the 14q12 region.
Rett syndrome is a severe neurodevelopment disorder and it is the second most common cause of mental
retardation in females. Beside the most common classic form, due to MECP2 mutations, a number of variants
have been observed such as the congenital variant. During my Ph.D program, I work in a project to study the
molecular cases of RETT Syndrome and By “candidate gene approach”, we have recently identified the new
gene FOXG1 (MIM 164874) as responsible for the congenital variant of RTT. FOXG1 gene encodes for a
brain-specific transcriptional repressor that bears three main functional domains: the fork-head domain
(FHD), the Groucho-binding domain (GTBD), and the JARID1B binding domain (JBD). After our first study,
mutations in the FOXG1 gene, located in 14q12 have been identified as the cause of this variant. Nineteen
cases have been reported to date with a FOXG1 loss of function alteration: seven deletions one duplication,
one inversion and ten point mutations. In collaboration with university of Freiburg we identified an additional
patient affected by the congenital variant of Rett having a whole FOXG1 deletion. We compared her clinical
features with the characteristics of patients already reported in the literature. Even though the reported cases
are still few and genotype phenotype correlation cannot be performed, there seems not to be a difference in
the clinical picture between deleted patients and patients with point mutations. This thesis work underlines
the importance to perform FOXG1 mutation analysis for both deletions and point mutations in patients with a
phenotype resembling the congenital variant of Rett, prior the analysis of the MECP2 gene.
This work is reported in:
Ariani et al. FOXG1 is responsible for the congenital variant of Rett syndrome. A. Am J Hum Genet. 2008
Jul;83(1):89-93.
Mencarelli et all. Novel FOXG1 mutations associated with the congenital variant of Rett syndrome. J Med Genet
2010;47:49e53. doi:10.1136/jmg.2009.067884
This work is funded by:
EMBO Fellowship Programme ASTF number 322-2010 and the University of Siena.
- 50 -
Oncology and Genetics Doctoral School
Oncology and Genetics Doctoral School
Hepatobiliopancreatic Disease and Multitumoral syndromes
XXIV cycle
Rosalia Zangari, BS
[email protected]
Tutor F. Cetta
The impact of traffic pollution on antioxidant system of two populations exposed to
different levels of pollutants
Aim of this study was to investigate whether the traffic pollution is related with alteration of thiol, redox and
energetic status, for the early recognition of at risk-subjects in a population exposed to environmental pollution, in a heavy polluted city, such as Milan, Italy.
We compared thiol levels, both in blood and in plasma, and erythrocyte redox (NADH, NAD+, NADPH AND
NADP+) and energetic (ATP, ADP and AMP) status in a group of subjects living downtown compared to a control population.
All analytes were assayed by HPLC. Thiols were measured both in reduced and total forms. Daily levels of
outdoor and indoor PM10, PM2.5 and PM1 concentrations were conducted using an optical particle counter
(OPC Grimm).
The concentrations within the building were heavily dependent on external concentration. During the day,
indoor PM concentration was higher than outdoor, but PM10 was the highest of all. This phenomenon is due
to a coarse particle resuspencion by internal domestic activity.
There was no evident difference in the GSH levels between the two populations. In fact, GSH alteration is
usually a late event, occurring in severe imbalances and no subject in both groups had clinically evident diseases. Inhabitants of areas with different traffic volumes show significantly different Cys CG and Hcy levels,
even if GSH remains unchanged, suggesting a greater- pro-oxidant effect in more exposed populations, affecting Cys CG and Hcy levels, before GSH alterations. A lower NADH level and a higher plasma total Hcy level
in more exposed populations is able to distinguish between more and less exposed subjects and could be a
useful diagnostic tool for early detection of subjects at higher risk of health effects from environmental pollution.
This work is reported in:
Zangari R. et al. The impact of traffic pollution on antioxidant system of two populations exposed to different levels of
pollutants: estimation of redox status of thiols.1286 “International Aerosol Conference (IAC 2010) Helsinki, Finland 29
August – 3 September 2010.
This work is funded by:
CARIPLO Grant to POLARIS and by the PROLIFE-Project, City of Milan, Italy.
- 51 -
- 52 -
Oncology and Genetics Doctoral School
Thesis discussion
Doctorate in Medical Genetics
February 5, 2010 Centro Didattico S Maria alle Scotte, room 15
10.00
Entering of the PhD dissertation board composed by:
- Prof. Maurizio Genuardi (President)
Professor of Medical Genetics, University of Firenze, Italy
- Prof. Emma D'Andrea (Member)
Professor of Pathology, University of Padova, Italy
- Prof. Giovanna Bianchi-Scarrà (Secretary)
Professor of Biology, University of Genova, Italy
Thesis discussion in English language:
- “Comprehensive analysis of the CDKN2A gene mutations in familial and multiple primary
cutaneous melanoma” , Marina Vignoli, XXI cycle
10.45 Awarding of the PhD degree in Medical Genetics
From left to right:
Prof. Giovanna Bianchi-Scarrà,
Prof. Maurizio Genuardi,
Marina Vignoli,
Prof. Emma D’Andrea.
- 53 -
Oncology and Genetics Doctoral School
Thesis discussion
Doctorate in
October 1, 2010 Centro Didattico S Maria alle Scotte, room 15
MORNING
9.00
SECTION
Entering of the PhD dissertation board composed by:
- Prof. Gabriello Tanzini (President)
Professor of Medical Surgery, University of Siena, Italy
- Prof. Pier Francesco Tassone (Member)
Professor of Medical Oncology, University of Catanzaro, Italy
- Prof. Generoso Bevilacqua (Member)
Professor of Human Pathology, University of Pisa, Italy
- Prof. Paola Mandich (Secretary)
Professor of Medical Genetics, University of Genova, Italy
Thesis discussion in English language:
- “Endothelin and colorectal cancer: information for prognosis and treatment” , Antonella Chessa, XXI cycle
9.45 Awarding of the PhD degree in Colorectal and Gastroesophageal Diseases
- 54 -
Oncology and Genetics Doctoral School
Thesis discussion
Doctorate in
October 1, 2010 Centro Didattico S Maria alle Scotte, room 15
10.00
Entering of the PhD dissertation board composed by:
- Prof. Alessandra Renieri (President)
Professor of Medical Genetics, University of Siena, Italy
- Prof. Paola Mandich (Member)
Professor of Medical Genetics, University of Genova, Italy
- Prof. Vaiditus Kucinskas (Member)
Human and Medical Genetics, Vilnius, Lithuania
- Prof. Mario Tecce (Secretary)
Professor of Biochemistry, University of Salerno, Italy
Thesis discussion in English language
- “Molecular work-up of patients with the congenital variant of Rett syndrome: analysis of the 14q12 region”,
Ariele Spanhol-Rosseto, XXII cycle
10.45 Awarding of the PhD degree in Medical Genetics and qualification of Doctor Europaeus
- 55 -
Oncology and Genetics Doctoral School
Thesis discussion
Doctorate in
October 1, 2010 Centro Didattico S Maria alle Scotte, room 15
11.00
Entering of the PhD dissertation board composed by:
- Prof. Alessandra Renieri (President)
Professor of Medical Genetics, University of Siena, Italy
- Prof. Paola Mandich (Member)
Professor of Medical Genetics, University of Genova, Italy
- Prof. Pier Francesco Tassone (Secretary)
Professor of Medical Oncology, University of Catanzaro, Italy
Thesis discussion in English language:
- “Autosomal Alport syndrome: a new model including both dominant and recessive inheritance” , Elena
Marcocci, XXII cycle
11.45 Awarding of the PhD degree in Medical Genetics
- 56 -
Oncology and Genetics Doctoral School
Thesis discussion
Doctorate in
October 1, 2010 Centro Didattico S Maria alle Scotte, room 15
12.00
Entering of the PhD dissertation board composed by:
- Prof. Alessandra Renieri (President)
Professor of Medical Genetics, University of Siena, Italy
- Prof. Mario Tecce (Member)
Professor of Biochemistry, University of Salerno, Italy
- Prof. Generoso Bevilacqua (Member)
Professor of Human Pathology, University of Pisa, Italy
- Prof. Paola Mandich (Secretary)
Professor of Medical Genetics, University of Genova, Italy
Thesis discussion in English language:
- “Retinoic acid and breast cancer: how to improve the therapeutic effect on the basis of molecular knowledge” , Valeria Guarnaccia, XXII cycle
12.45 Awarding of the PhD degree in Medical Genetics
- 57 -
Oncology and Genetics Doctoral School
Thesis discussion
Doctorate in
October 1, 2010 Centro Didattico S Maria alle Scotte, room 15
AFTERNOON
14.00
SECTION
Entering of the PhD dissertation board composed by:
- Prof. Antonio Giordano (President)
Professor of Human Pathology, University of Siena, Italy
- Prof. Pier Francesco Tassone (Member)
Professor of Medical Oncology, University of Catanzaro, Italy
- Prof. Generoso Bevilacqua (Member)
Professor of Human Pathology, University of Pisa, Italy
- Prof. Wolfgang Bohn (Member)
Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg, Germany
- Prof. Mario Tecce (Secretary)
Professor of Biochemistry, University of Salerno, Italy
Thesis discussion in English language
- “A Phase 1 Dose Escalation Study of ARQ 197 in Adult Patients with Metastatic Solid Tumors” ,
Abbadessa Giovanni, XXII cycle
14.45 Awarding of the PhD degree in Oncological Genetics and qualification of Doctor Europaeus
- 58 -
Oncology and Genetics Doctoral School
Thesis discussion
Doctorate in
October 1, 2010 Centro Didattico S Maria alle Scotte, room 15
15.00
Entering of the PhD dissertation board composed by:
- Prof. Antonio Giordano (President)
Professor of Human Pathology, University of Siena, Italy
- Prof. Pier Francesco Tassone (Member)
Professor of Medical Oncology, University of Catanzaro, Italy
- Prof. Generoso Bevilacqua (Member)
Professor of Human Pathology, University of Pisa, Italy
- Prof. Wolfgang Bohn (Member)
Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg, Germany
- Prof. Mario Tecce (Secretary)
Professor of Biochemistry, University of Salerno, Italy
Thesis discussion in English language:
- “miRNA expression profiling in malignant mesothelioma” , Khadang Baharak, XXII cycle
15.45 Awarding of the PhD degree in Oncological Genetics and qualification of Doctor Europaeus
- 59 -
Oncology and Genetics Doctoral School
Thesis discussion
Doctorate in
October 1, 2010 Centro Didattico S Maria alle Scotte, room 15
16.00
Entering of the PhD dissertation board composed by:
- Prof. Antonio Giordano (President)
Professor of Human Pathology, University of Siena, Italy
- Prof. Pier Francesco Tassone (Member)
Professor of Medical Oncology, University of Catanzaro, Italy
- Prof. Generoso Bevilacqua (Member)
Professor of Human Pathology, University of Pisa, Italy
- Prof. Wolfgang Bohn (Member)
Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg, Germany
- Prof. Mario Tecce (Secretary)
Professor of Biochemistry, University of Salerno, Italy
Thesis discussion in English language:
- “PIN1, the cell cycle control and cancer: a new player in the RB pathway” , Flavio Rizzolio, XXII cycle
16.45 Awarding of the PhD degree in Oncological Genetics and qualification of Doctor Europaeus
- 60 -
Oncology and Genetics Doctoral School
Thesis discussion
Doctorate in
December 14, 2010 Centro Didattico S Maria alle Scotte, room 15
MORNING
9.00
SECTION
Entering of the PhD dissertation board composed by:
- Prof. Alessandra Renieri (President)
Professor of Medical Genetics, University of Siena, Italy
- Prof. Mario De Marchi (Member)
Professor of Medical Genetics, University of Torino, Italy
- Prof. Francesca Gensini (Secretary)
Professor of Medical Genetics, University of Firenze, Italy
Thesis discussion in English language:
- “Diagnosis process assessment of Array-CGH analysis” , Filomena Tiziana Papa, XXII cycle
9.45 Awarding of the PhD degree in Medical Genetics
- 61 -
Oncology and Genetics Doctoral School
Thesis discussion
Doctorate in
December 14, 2010 Centro Didattico S Maria alle Scotte, room 15
10.00
Entering of the PhD dissertation board composed by:
- Prof. Francesco Cetta (President)
Professor of Medical Surgery, University of Siena, Italy
- Prof. Mario De Marchi (Member)
Professor of Medical Genetics, University of Torino, Italy
- Prof. Francesca Gensini (Secretary)
Professor of Medical Genetics, University of Firenze, Italy
Thesis discussion in English language:
- “Correlation between aging and tumorigenesis: the role of genetic factors in Werner syndrome and related
syndromes” , Simona Benoni, XXII cycle
10.45 Awarding of the PhD degree in Hepatobiliopancreatic Diseases and Multitumoral Syndromes
- 62 -
Oncology and Genetics Doctoral School
Thesis discussion
Doctorate in
December 14, 2010 Centro Didattico S Maria alle Scotte, room 15
11.00
Entering of the PhD dissertation board composed by:
- Prof. Francesco Cetta (President)
Professor of Medical Surgery, University of Siena, Italy
- Prof. Mario De Marchi (Member)
Professor of Medical Genetics, University of Torino, Italy
- Prof. Francesca Gensini (Secretary)
Professor of Medical Genetics, University of Firenze, Italy
Thesis discussion in English language:
- “Role of diet and gender in the onset and/or protection in tumors” , Giuliana Malagnino, XXI cycle
11.45 Awarding of the PhD degree in Hepatobiliopancreatic Diseases and Multitumoral Syndromes
- 63 -
Oncology and Genetics Doctoral School
Thesis discussion
Doctorate in
December 14, 2010 Centro Didattico S Maria alle Scotte, room 15
12.00
Entering of the PhD dissertation board composed by:
- Prof. Francesco Cetta (President)
Professor of Medical Surgery, University of Siena, Italy
- Prof. Mario De Marchi (Member)
Professor of Medical Genetics, University of Torino, Italy
- Prof. Francesca Gensini (Secretary)
Professor of Medical Genetics, University of Firenze, Italy
Thesis discussion in English language:
- “Genetics and molecular biology in the multidisciplinary approach to pressure ulcers and lower limb ulcers
in diabetics” , Cisternino Filomena, XXII cycle
12.45 Awarding of the PhD degree in Hepatobiliopancreatic Diseases and Multitumoral Syndromes
- 64 -
Oncology and Genetics Doctoral School
Thesis discussion
Doctorate in
December 14, 2010 Centro Didattico S Maria alle Scotte, room 15
AFTERNOON
14.00
SECTION
Entering of the PhD dissertation board composed by:
- Prof. Francesco Cetta (President)
Professor of Medical Surgery, University of Siena, Italy
- Prof. Mario De Marchi (Member)
Professor of Medical Genetics, University of Torino, Italy
- Prof. Francesca Gensini (Secretary)
Professor of Medical Genetics, University of Firenze, Italy
Thesis discussion in English language:
- “Papillary carcinoma of thyroid associated to familial adenomatosis” , Barellini Leonardo , XX cycle
14.45 Awarding of the PhD degree in Hepatobiliopancreatic Diseases and Multitumoral Syndromes
- 65 -
Oncology and Genetics Doctoral School
Thesis discussion
Doctorate in
December 14, 2010 Centro Didattico S Maria alle Scotte, room 15
15.00
Entering of the PhD dissertation board composed by:
- Prof. Francesco Cetta (President)
Professor of Medical Surgery, University of Siena, Italy
- Prof. Mario De Marchi (Member)
Professor of Medical Genetics, University of Torino, Italy
- Prof. Francesca Gensini (Secretary)
Professor of Medical Genetics, University of Firenze, Italy
Thesis discussion in English language:
- “Predictors of invasivity in Intraductal Pancreatic Mucinous Neoplasms” , Mariani Federico , XIX cycle
15.45 Awarding of the PhD degree in Hepatobiliopancreatic Diseases and Multitumoral Syndromes
- 66 -